Dig1/Dig2/Ste12/Tec1 interactions

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Contents

General Notes

Dig1/Dig2/Ste12 Complex

  • Over-expression of Ste12 by itself is lethal, whereas simultaneous over-expression of either Dig1 or Dig2 restores viability. Tedford et al. 1997 PMID 9094309
  • Expression of a fusion between the Gal4 activation domain and Dig1 or Dig2 results in constitutive expression of FUS1-lacZ, as well as a constitutive invasive growth phenotype. Tedford et al. 1997 PMID 9094309
    • This effect is Ste12 dependent.
    • This suggests that Dig1 and Dig2 bind Ste12 and co-localize with Ste12 to pheromone and filamentous response elements (PREs and FREs).
  • Mutation of Y310 and/or Y317 to alanine in Ste12(301-335) of GBD-Ste12(301-335)GADI (Gal4 DNA binding domain fused to Ste12(301-335) fused to Gal4 activation domain I) increases transcriptional activity in the absence of pheromone, and virtually no increase in transcription in the presence of pheromone. Pi et al. 1997 PMID 9343403
    • These mutations eliminate two-hybrid interactions between Ste12(301-335) and Dig1 and Dig2, suggesting they are important for Dig1 and Dig2 binding to Ste12.
    • Ste12(Y310A Y317A) has 6x higher FUS1-lacZ expression than WT Ste12 in the absence of pheromone, but the same expression in the presence of pheromone.
    • This suggests that Y310 and Y317 are only responsible for part of the pheromone induced derepression and activation of Ste12.
    • Olson et al. (2000 PMID 10825185) suggest that the two-hybrid interaction between Ste12(301-335) and Dig1 may be as result of Ste12 dimerization by the Gla4 DBD (see below).
    • Olson et al. suggest that the two-hybrid interaction observed between, Ste12(301-335) and Dig2 may be as result of interaction with WT Ste12 that is in the cell (though exactly how this would work remains unclear because Ste12(301-335) fusion protein presumably contains no means of multimerizing with WT Ste12).
  • Using proteins generated by in vitro transcription/translation, myc-Ste12 pulls down a small amount of Dig1 or Dig2 when present separately (1:0.3 and 1:0.2 stoichiometries, respectively), but a large amount of both Dig1 and Dig2 when they are present together (Ste12:Dig1:Dig2=1:1.4:1.5). Chou et al. 2006 PMID 16782869
  • Deletion of Dig1 has little effect on the amount of Dig2 found at the PREs of FUS1 (measured by ChIP). Chou et al. 2006 PMID 16782869
    • This suggests that either Dig1 and Dig2 do not bind cooperatively to Ste12 (or maybe not while Ste12 is bound to DNA), or the physiological amount of Dig2 in the cells is high enough to saturate Ste12 even in the absence of Dig1.
  • Overexpression of Ste12 (and to a lesser extent Ste12(262-594)) in WT cells results in elevated FUS1-lacZ expression. Olson et al. 2000 PMID 10825185
    • Since Ste12(262-594) does not bind Dig2, the mechanism of activation is likely not through titration of the repressor proteins Dig1 and Dig2 in order to de-repress native Ste12.
    • When Ste12(262-594) is overexpressed in WT cells, deletion of either Dig1 or Dig2 results in higher FUS1 expression than in WT cells, even though only Dig1 should bind to this fragment.

Dig1/Ste12/Tec1 Complex

  • Ste12 association with mating-specific promoters in response to pheromone is largely eliminated by deletion of Fus3 and Kss1, whereas Ste12 association with filamentation-specific promoters (again in response to pheromone) is largely unchanged by deletion of Fus3 and Kss1 (measured by ChIP). Zeitlinger et al. 2003 PMID 12732146
    • This suggests that even while Ste12 is highly repressed by Dig1 and Dig2 in fus3Δ kss1Δ cells (such that it is unable to efficiently bind mating promoters), Ste12 is still able to associate with filamentous promoters, presumably through Ste12's association with Tec1.
  • Tec1 appears to interact with Ste12 via residues 281-486. Bruckner et al. 2004 PMID 15558284
    • Tec1 co-purifies with GST-Kss1 in WT cells, but not in ste12Δ cells. This suggests that Kss1 can only associate with Tec1 via Ste12.
    • Tec1(1-280) does not co-purify with GST-Kss1 in WT cells, but Tec1(281-486) does co-purify with GST-Kss1 in WT cells. This suggests that Tec1 may interact with Ste12 via its C-terminal half.
  • Increasing amounts of Tec1 affect the amount of Dig2 that myc-Ste12 can pull down (using proteins generated by in vitro transcription/translation). Chou et al. 2006 PMID 16782869
    • The amount of Dig1 that associated with Ste12 also decreases with increasing amounts of Tec1, consistent with Dig1 and Dig2 binding cooperatively to Ste12.
    • When the same experiment is performed in the absence of Dig2, the amount of Dig1 that associated with Ste12 is unaffected by increasing amounts of Tec1.
    • When Tec1 is overexpressed in cells from the GAL1 promoter, much less Dig2-myc coimmunoprecipitates with Ste12-HA.
  • Tec1-HA is able to immunoprecipitate Dig1-myc only when Ste12 is also present in the cell, demonstrating that Dig1's interaction with Tec1 (and thus repression of Tec1 activity) is through Ste12. Chou et al. 2006 PMID 16782869
  • Residues 300-400 of Tec1 are necessary (although not sufficient) for it's interaction with Ste12 (immunoprecipitation). Chou et al. 2006 PMID 16782869
    • Deletions that eliminate Tec1's interaction with Ste12 also eliminate LexA-Tec1 mediated gene expression in Dig1Δ cells, suggesting that Ste12 is responsible for the transcriptional activity associated with Tec1.
  • Expression off of TCS-LacZ and FRE-LacZ is elevated upon deletion of Dig1, but not deletion of Dig2. Simultaneous deletion of Dig1 and Dig2 results in expression that is moderately higher than deletion of Dig1 by itself. Chou et al. 2006 PMID 16782869
    • This suggests that Dig1 regulates expression of filamentous genes, and Dig2 does not.
  • Expression mediated by a fusion of the DNA binding domain of LexA with Tec1 is repressed by Dig1, but not by Dig2, demonstrating that Dig1 alone acts to repress Tec1 activity. Chou et al. 2006 PMID 16782869
  • Using ChIP, nearly equal amounts of Ste12-myc, Tec1-myc and Dig1-myc were found to be located at TCS in filamentous gene promoters. Dig2-myc was detected at the promoters at very low levels. Chou et al. 2006 PMID 16782869
  • Using ChIP, nearly equal amounts of Ste12-myc, and Dig1-myc were found to be located at TCS in filamentous gene promoters. Dig2-myc was detected at the promoters at slightly lower levels (consistent with its potential role of inhibiting Ste12's interaction with PREs), and Tec1-myc was detected at the promoters at very low levels. Chou et al. 2006 PMID 16782869

Dig1/Ste12 Interactions

  • Dig1 binds Ste12(309-547). Olson et al. 2000 PMID 10825185
    • GST-Dig1 binds Ste12(309-547), but GST-Dig2 does not.
    • TRPE-Ste12(216-688) expressed in E. coli can be affinity precipitated by purified GST-Dig1, suggesting that this interaction is direct.
    • Dig1 (but not Dig2) can repress FUS1-lacZ expression mediated by LexA-Ste12(216-688) in the absence of pheromone.
    • Both Dig1 and Dig2 can repress FUS1-lacZ expression mediated by LexA-Ste12.
  • By ChIP, Dig1 was found to associate with the same set of promoters as Ste12 (pheromone responsive and filamentous growth promoters), suggesting that Dig1 can remain associated with Ste12 while Ste12 is bound to DNA. Zeitlinger et al. 2003 PMID 12732146
  • Dig1 may bind to Ste12 more strongly in the context of Ste12 multimers.
    • Pi et al. (1997 PMID 9343403) showed that LexA-Ste12(301-335) is able to interact with Dig1 (fused to Gal4 activating domain) via two-hybrid.
    • Pi et al. also showed that a fusion protein with Ste12(301-335) fused to the Gal4 DNA binding domain and the Gal4 transcriptional activation domain I (GBD-Ste12(301-335)GADI) is activated in response to pheromone, and this effect is eliminated by fus3Δ kss1Δ or ste5Δ mutants, suggesting that Ste12(301-335) is repressible by at least one of Dig1 or Dig2.
    • Olson et al. (2000 PMID 10825185) showed that Ste12(309-547) (and no smaller fragment) bound GST-Dig1.
    • Olson et al. suggest that Ste12 multimerization may play an important role in Dig1 binding because in their case the minimal Ste12 fragment contains the multimerization domain. Though Olson et al. mention only that the Gal4 DNA binding domain forms a dimer, it appears that LexA (used by Pi et al. fused to Ste12 for their two-hybrid) is known to dimerize and thus might cause Ste12 multimerization.
    • Olson et al. showed that GST-Dig1 binds Gal4-Ste12(216-473), but it does not bind Ste12(216-500), indicating that the Gal4 is playing a role.

Reaction Definition

Assumptions:

  • Dig1 binding to Ste12 is unaffected by dimerization/multimerization of Ste12.
    • Evidence that Dig1 binds preferably to a Ste12 dimer is circumstantial (see arguments from Olson et al. above).
    • Chou et al. show that the stoichiometry of Ste12:Dig1:Dig2 complexes in vitro is 1:1.4:1.5, which suggests that a single Dig1 does not bind to multiple Ste12 monomers, but rather binds Ste12 in a 1:1 ratio.
Ste12(Dig1_site, Dig2_site, MAPK_site) + Dig1(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!1, Dig2_site, MAPK_site).Dig1(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site, MAPK_site) + Dig1(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!1, Dig2_site, MAPK_site).Dig1(Ste12_site!1, PO4_site~PO4)


Ste12(Dig1_site, Dig2_site!+, MAPK_site) + Dig1(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!1, Dig2_site!+, MAPK_site).Dig1(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site!+, MAPK_site) + Dig1(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!1, Dig2_site!+, MAPK_site).Dig1(Ste12_site!1, PO4_site~PO4)


Ste12(Dig1_site, Dig2_site, MAPK_site!2).Kss1(docking_site!2) + Dig1(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!1, Dig2_site, MAPK_site!2).Kss1(docking_site!2).Dig1(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site, MAPK_site!2).Kss1(docking_site!2) + Dig1(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!1, Dig2_site, MAPK_site!2).Kss1(docking_site!2).Dig1(Ste12_site!1, PO4_site~PO4)


Ste12(Dig1_site, Dig2_site!+, MAPK_site!2).Kss1(docking_site!2) + Dig1(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!1, Dig2_site!+, MAPK_site!2).Kss1(docking_site!2).Dig1(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site!+, MAPK_site!2).Kss1(docking_site!2) + Dig1(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!1, Dig2_site!+, MAPK_site!2).Kss1(docking_site!2).Dig1(Ste12_site!1, PO4_site~PO4)


Ste12(Dig1_site, Dig2_site, MAPK_site!2).Fus3(docking_site!2) + Dig1(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!1, Dig2_site, MAPK_site!2).Fus3(docking_site!2).Dig1(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site, MAPK_site!2).Fus3(docking_site!2) + Dig1(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!1, Dig2_site, MAPK_site!2).Fus3(docking_site!2).Dig1(Ste12_site!1, PO4_site~PO4)


Ste12(Dig1_site, Dig2_site!+, MAPK_site!2).Fus3(docking_site!2) + Dig1(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!1, Dig2_site!+, MAPK_site!2).Fus3(docking_site!2).Dig1(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site!+, MAPK_site!2).Fus3(docking_site!2) + Dig1(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!1, Dig2_site!+, MAPK_site!2).Fus3(docking_site!2).Dig1(Ste12_site!1, PO4_site~PO4)


See Dig1/Dig2/Ste12/MAPK binding rate constant constraints.

Dig2/Ste12 Interactions

  • Dig2 binds Ste12(21-195), which is part of Ste12's DNA binding domain. Olson et al. 2000 PMID 10825185
    • GST-Dig2 binds His6-Ste12(21-195), but GST-Dig1 does not.
    • Dig2 (but not Dig1) can repress FUS1-lacZ expression mediated by a fusion between the DNA binding domain of Ste12 (Ste12(1-215)) and the transcriptional activation domain of the herpes simplex virus type 1 (VP16), suggesting that Dig2 acts to interfere with Ste12 DNA binding.
    • Either Dig1 or Dig2 alone is sufficient to repress FUS1-LacZ expression mediated by LexA-Ste12.

Reaction Definition

Assumptions:

  • Although Dig2 binding to Ste12 is stabilized by Kss1 or Fus3 binding to Ste12, we will assume that the MAPK actually only stabilizes Dig1 binding to Ste12. Dig2 binding to Ste12 is indirectly stabilized due to the cooperativity between Dig1 and Dig2 binding to Ste12 (see Kss1/Ste12 interactions section below for more).
  • Dig2 binding to Ste12 is unaffected by dimerization/multimerization of Ste12.
    • Unlike Dig1, there is no evidence at all (even circumstantial) suggesting that Dig2 binds preferably to a Ste12 dimer.
    • Chou et al. show that the stoichiometry of Ste12:Dig1:Dig2 complexes in vitro is 1:1.4:1.5, which suggests that a single Dig2 does not bind to multiple Ste12 monomers, but rather binds Ste12 in a 1:1 ratio.
Ste12(Dig1_site, Dig2_site) + Dig2(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site, Dig2_site!1).Dig2(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site, Dig2_site) + Dig2(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site, Dig2_site!1).Dig2(Ste12_site!1, PO4_site~PO4)


Ste12(Dig1_site!+, Dig2_site) + Dig2(Ste12_site, PO4_site~none) <->

Ste12(Dig1_site!+, Dig2_site!1).Dig2(Ste12_site!1, PO4_site~none)


Ste12(Dig1_site!+, Dig2_site) + Dig2(Ste12_site, PO4_site~PO4) <->

Ste12(Dig1_site!+, Dig2_site!1).Dig2(Ste12_site!1, PO4_site~PO4)


See Dig1/Dig2/Ste12/MAPK binding rate constant constraints.

Kss1/Ste12 Interactions

  • Phosphorylation on Kss1's activation loop is necessary and sufficient to relieve Kss1-dependent inhibition of invasive growth. Bardwell et al. 1998 PMID 9744865
    • Unphosphorylatable Kss1 mutants (T183A, Y185F, and T183A Y185F) prevent invasive growth in fus3Δ kss1Δ cells.
    • Catalytically inactive Kss1 mutants (Y24F and K42R Q45P) allow for invasive growth in fus3Δ kss1Δ cells.
    • Transcription off of FRE's also mimics results from invasive growth assays.
  • Deletion of Dig1 and Dig2, or Fus3 and Kss1 results in a hyperinvasive phenotype. Overexpression of Dig1 represses transcription off of FREs in Fus3Δ Kss1Δ cells, whereas overexpression of Kss1 does not repress transcription off of FREs in Dig1Δ Dig2Δ cells. Bardwell et al. 1998 PMID 9860980
  • Inactive Kss1 acts as a stronger transcriptional repressor at FREs than at PREs. Bardwell et al. 1998 PMID 9860980
    • In Dig1/Dig2 mediated repression of PREs, deletion of Kss1 results in a moderate (7-fold) increase FUS1 gene expression in the absence of pheromone, but this level is only 7% of the pheromone induced level.
    • In Dig1/Dig2 mediated repression of FREs, deletion of Kss1 (in ste7Δ cells, thus no signal) results in a 60% of the FRE expression observed in normal invasive cells. Deletion of Fus3 raises this to 100%.
  • Unphosphorylated Kss1 interacts with Ste12 with a Kd of ~ 400 nM (determined by cosedimentation). Bardwell et al. 1998 PMID 9744865
    • Mutations to the activation loop on Kss1 eliminate its interaction with Ste12, but not with Ste7, Dig1 and Dig2 (via two-hybrid, in vitro binding, and coimmunoprecipitation).
    • Mutations in Kss1's activation loop which selectively prevent binding to Ste12 cause constitutive invasive growth and FRE-mediated transcription in ste7Δ kss1Δ fus3Δ cells.
    • Phosphorylated Kss1 interacts much more weakly with Ste12 than unphosphorylated Kss1.

Reaction Definition

Assumptions:

Ste12(Dig1_site, MAPK_site) + Kss1(docking_site, T183~none, Y185~none) <->

Ste12(Dig1_site, MAPK_site!1).Kss1(docking_site!1, T183~none, Y185~none)


Ste12(Dig1_site, MAPK_site) + Kss1(docking_site, T183~PO4, Y185~none) <->

Ste12(Dig1_site, MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~none)


Ste12(Dig1_site, MAPK_site) + Kss1(docking_site, T183~none, Y185~PO4) <->

Ste12(Dig1_site, MAPK_site!1).Kss1(docking_site!1, T183~none, Y185~PO4)


Ste12(Dig1_site, MAPK_site) + Kss1(docking_site, T183~PO4, Y185~PO4) <->

Ste12(Dig1_site, MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~PO4)


Ste12(Dig1_site!+, MAPK_site) + Kss1(docking_site, T183~none, Y185~none) <->

Ste12(Dig1_site!+, MAPK_site!1).Kss1(docking_site!1, T183~none, Y185~none)


Ste12(Dig1_site!+, MAPK_site) + Kss1(docking_site, T183~PO4, Y185~none) <->

Ste12(Dig1_site!+, MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~none)


Ste12(Dig1_site!+, MAPK_site) + Kss1(docking_site, T183~none, Y185~PO4) <->

Ste12(Dig1_site!+, MAPK_site!1).Kss1(docking_site!1, T183~none, Y185~PO4)


Ste12(Dig1_site!+, MAPK_site) + Kss1(docking_site, T183~PO4, Y185~PO4) <->

Ste12(Dig1_site!+, MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~PO4)


See Dig1/Dig2/Ste12/MAPK binding rate constant constraints.

Fus3/Ste12 Interactions

  • Fus3 binding to GST-Ste12(298-473) is not detectable (whereas Kss1 binding is), and Fus3 shows a weaker association with Ste12 than Kss1 in yeast cell extracts. Bardwell et al. 1998 PMID 9744865

Reaction Definition

Assumptions:

  • Phosphorylation of Fus3 decreases its affinity for Ste12.
Ste12(Dig1_site, MAPK_site) + Fus3(docking_site, T180~none, Y182~none) <->

Ste12(Dig1_site, MAPK_site!1).Fus3(docking_site!1, T180~none, Y182~none)


Ste12(Dig1_site, MAPK_site) + Fus3(docking_site, T180~PO4, Y182~none) <->

Ste12(Dig1_site, MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~none)


Ste12(Dig1_site, MAPK_site) + Fus3(docking_site, T180~none, Y182~PO4) <->

Ste12(Dig1_site, MAPK_site!1).Fus3(docking_site!1, T180~none, Y182~PO4)


Ste12(Dig1_site, MAPK_site) + Fus3(docking_site, T180~PO4, Y182~PO4) <->

Ste12(Dig1_site, MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~PO4)


Ste12(Dig1_site!+, MAPK_site) + Fus3(docking_site, T180~none, Y182~none) <->

Ste12(Dig1_site!+, MAPK_site!1).Fus3(docking_site!1, T180~none, Y182~none)


Ste12(Dig1_site!+, MAPK_site) + Fus3(docking_site, T180~PO4, Y182~none) <->

Ste12(Dig1_site!+, MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~none)


Ste12(Dig1_site!+, MAPK_site) + Fus3(docking_site, T180~none, Y182~PO4) <->

Ste12(Dig1_site!+, MAPK_site!1).Fus3(docking_site!1, T180~none, Y182~PO4)


Ste12(Dig1_site!+, MAPK_site) + Fus3(docking_site, T180~PO4, Y182~PO4) <->

Ste12(Dig1_site!+, MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~PO4)


See Dig1/Dig2/Ste12/MAPK binding rate constant constraints.

Ste12/Tec1 Interactions

We will not model Tec1, and filamentation gene expression.

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